Phycobiliproteins, fucoxanthin, and krill oil are natural marine products with excellent activities. In the study, we prepared the complex of phycobiliproteins, fucoxanthin, and krill oil (PFK) and assessed the anti-obesity, lipid-lowering, and antioxidant activities in high-fat diet rats. The results showed that the rats significantly and safely reduced body weight gain and regulated serum biochemical parameters at 50 mg/kg phycobiliproteins, 10 mg/kg fucoxanthin, and 100 mg/kg krill oil. Furthermore, the molecular mechanism study suggested that the complex of PFK confined the enzyme activities of lipid synthesis and enhanced antioxidant activity to improve obesity indirectly. The conclusions demonstrated that the complex of PFK has potent anti-obesity and hypolipidemic effects which have potential use as a natural and healthy food and medicine for anti-obesity and lowering blood lipids in the future.

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.

ABSTRACT Simian hemorrhagic fever virus (SHFV) was recently reclassified and assigned to the new virus family Arteriviridae . During replication, arteriviruses produce a 3′ coterminal, nested set of subgenomic mRNAs (sgRNAs). These sgRNAs arise by discontinuous transcription, and each contains a 5′ leader sequence which is joined to the body of the mRNA through a conserved junction sequence. Only the 5′-most open reading frame (ORF) is believed to be transcribed from each sgRNA. The SHFV genome encodes nine ORFs that are presumed to be expressed from sgRNAs. However, reverse transcription-PCR analysis with leader- and ORF-specific primers identified only eight sgRNA species. The consensus sequence 5′-UCNUUAACC-3′ was identified as the junction motif. Our data suggest that sgRNA 2 may be bicistronic, expressing both ORF 2a and ORF 2b. SHFV encodes three more ORFs on its genome than the other arteriviruses. Comparative sequence analysis suggested that SHFV ORFs 2a, 2b, and 3 are related to ORFs 2 through 4 of the other arteriviruses. Evidence which suggests that SHFV ORFs 4 through 6 are related to ORFs 2a through 3 and may have resulted from a recombination event during virus evolution is presented.

Sero-epidemiological surveys have revealed that equine arteritis virus (EAV) is prevalent in most European countries. The virus causes sporadic cases of respiratory disease and abortion in horses, the incidence of which has increased in recent years. Mares and geldings eliminate virus after acute infection, but 30% to 60% of stallions become persistently infected. In these animals, EAV is maintained within the reproductive tract and is shed continuously in the semen. Persistent infection with EAV in stallions has no negative consequences for fertility but mares inseminated with virus-contaminated semen can have an acute infection. These mares shed large amounts of virus in respiratory secretions and urine, leading to lateral spread of the virus to other susceptible horses. Acute infection at later stages of gestation can lead to abortion. Effective control of the spread of EAV infection depends on the identification of virus-shedding stallions. Persistently infected stallions should not be used for breeding or should be bred only to seropositive mares. Mares bred to shedding stallions should be isolated from other animals for a period of 3 weeks following insemination to prevent the lateral spread of EAV.

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae. which can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Mares and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of greater than or equal to 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating. Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination; the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen. (C) A.L. Glaser at al. 1997.

Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.

The small membrane glycoprotein (GS) of equine arteritis virus (EAV) is a minor virion component but is abundantly expressed in EAV-infected cells. In this study, we have analyzed its membrane topology, folding, oligomerization, and intracellular transport. We show that GS is a class I integral membrane protein with one functional N-glycosylation site. Gel electrophoresis under nonreducing conditions revealed that GS occurs in EAV-infected cells in four monomeric conformations and as disulfide-linked homodimers. The slowest-migrating monomeric form corresponded to the fully reduced GS protein; the three faster-migrating monomeric species are probably generated by the formation of alternative intrachain disulfide bonds between the three luminal cysteines in the molecule. The GS monomers were selectively retained in the endoplasmic reticulum, as judged by their permanent susceptibility to endoglycosidase H, whereas the GS dimers were specifically incorporated into virus particles and became endoglycosidase H resistant and sialylated during passage through the Golgi apparatus.

In a coimmunoprecipitation assay with monospecific antisera, the two major envelope proteins GL and M of equine arteritis virus were found to occur in heteromeric complexes in virions and infected cells. While the GL protein associated with M rapidly and efficiently, newly synthesized M protein was incorporated into complexes at a slower rate, which implies that it interacts with GL molecules synthesized earlier. Analysis under nonreducing conditions revealed that the GL/M complexes consist of disulfide-linked heterodimeric structures. Pulse-chase experiments showed that virtually all GL monomers ended up in heterodimers, whereas a fraction of the M protein persisted as monomers. The M protein also formed covalently linked homodimers, but only the heterodimers were incorporated into virus particles.

Complementary DNAs encoding ORFs 2 to 7 of equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (G(L)) was identified as the most reactive to EAV-positive equine sera and an immunodominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a G(L)-specific synthetic peptide (residues 75 through 97) induced EAV-neutralizing antibody in vaccinated horses. The defined antigenic region of G(L) is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

Equine arteritis virus (EAV) belongs to a group of small enveloped positive-stranded RNA viruses, provisionally designated arteriviruses, which further includes lactate dehydrogenase-elevating virus (LDV), simian hemorrhagic fever virus (SHFV), and swine infertility and respiratory syndrome virus (SIRSV) or Lelystad virus (LV). These viruses resemble togaviruses in their physicochemical properties, the polarity and size (13–15 kb) of their genomes, and the size (50–70 nm) and morphology (isometric) of the virionsl,2 but virus assembly occurs by intracellular budding1,3. In contrast, the genome organization and gene expression strategy are similar to those of corona-and toroviruses2,4,5,6,7,8.

We have recently shown that the genome of equine arteritis virus (EAV) contains seven open reading frames (ORFs). We now present data on the structural proteins of EAV and the assignment of their respective genes. Virions are composed of a 14-kDa nucleocapsid protein (N) and three membrane proteins designated M, GS, and GL. M is an unglycosylated protein of 16 kDa, and GS and GL are N-glycosylated proteins of 25 and 30 to 42 kDa, respectively. The broad size distribution of GL results from heterogeneous N-acetyllactosamine addition since it is susceptible to digestion by endo-beta-galactosidase. Using monospecific antisera as well as an antivirion serum, and by expression of individual ORFs, the genes for the structural proteins were identified: ORF 7 codes for N, ORF 6 for M, ORF 5 for GL, and ORF 2 for GS. With the exception of GS, the proteins are about equally abundant in EAV virions, being present at a molar ratio of 3 (N):2 (M):3 (GL). The GS protein, which is expressed at a level similar to that of M in infected cells, is strikingly underrepresented in virus particles (1 to 2%). Our data justify a distinct taxonomic position for EAV, together with lactate dehydrogenase-elevating virus and simian hemorrhagic fever virus; although coronavirus- and toroviruslike in features of transcription and translation, the virion architecture of EAV is fundamentally different.

The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily.

During the replication of equine arteritis virus (EAV) six subgenomic mRNAs are synthesized. We present evidence that the viral mRNAs form a 3'-coterminal nested set and contain a common leader sequence of 208 nucleotides which is encoded by the 5'-end of the genome. The leader is joined to the bodies of mRNA 5 and 6 at positions defined by the sequence 5' UCAAC 3'. The part of the leader sequence flanking the UCAAC motif is very similar to the 5'-splice site of the Tetrahymena pre-rRNA. A possible internal guide sequence has been identified 43 nucleotides downstream of the leader sequence on the genome. Hybridization analysis shows that all EAV intracellular RNAs contain the leader sequence. These data imply that the viral subgenomic mRNAs are composed of leader and body sequences which are non-contiguous on the genome.